The success of various synthetic nucleosides such as AZT, D4T, DDI, and DDC in inhibiting the replication of HIV in vivo or in vitro led researchers in the late 1980""s to design and test nucleosides that substitute a heteroatom for the carbon atom at the 3xe2x80x2-position of the nucleoside. Norbeck, et al., disclosed that (xc2x1)-1-[cis-(2,4)-2-(hydroxymethyl)-4-dioxolanyl]thymine (referred to as (xc2x1)-dioxolane-T) exhibits a modest activity against HIV (EC50 of 20 xcexcM in ATH8 cells), and is not toxic to uninfected control cells at a concentration of 200 xcexcM. Tetrahedron Letters 30 (46), 6246, (1989). European Patent Application Publication No. 337 713 and U.S. Pat. No. 5,041,449, assigned to BioChem Pharma, Inc., disclose racemic 2-substituted-4-substituted-1,3-dioxolanes that exhibit antiviral activity. Published PCT application numbers PCT US91/09124 and PCT US93/08044 disclose isolated xcex2-D-1,3-dioxolanyl nucleosides for the treatment of HIV infection. WO 94/09793 discloses the use of isolated xcex2-D-1,3-dioxolanyl nucleosides for the treatment of HBV infection.
Published PCT US95/11464 discloses that (xe2x88x92)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine is useful in the treatment of tumors and other abnormal cell proliferation.
U.S. Pat. No. 5,047,407 and European Patent Application Publication No. 0 382 526, also assigned to BioChem Pharma, Inc., disclose that a number of racemic 2-substituted-5-substituted-1,3-oxathiolane nucleosides have antiviral activity, and specifically report that the racemic mixture of 2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (referred to below as BCH-189) has approximately the same activity against HIV as AZT, with less toxicity. The (xe2x88x92)-enantiomer of BCH-189 (U.S. Pat. No. 5,539,116 to Liotta, et al.), known as 3TC, is now sold commercially for the treatment of HIV in humans in the United States. See also EP 513 200 B1.
It has also been disclosed that cis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (xe2x80x9cFTCxe2x80x9d) has potent HIV activity. See Schinazi, et al., xe2x80x9cSelective Inhibition of Human Immunodeficiency viruses by Racemates and Enantiomers of cis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-Oxathiolane-5-yl]Cytosinexe2x80x9d Antimicrobial Agents and Chemotherapy, November 1992, page 2423-2431. See also U.S. Pat. Nos. 5,814,639; 5,914,331; 5,210,085; U.S. Pat. No. 5,204,466, WO 91/11186, and WO 92/14743.
Because of the commercial importance of 1,3-oxathiolane nucleosides, a number of processes for their production have been described in patents and scientific literature. Three key aspects of the synthesis must be considered during design of the process. First, the reaction scheme must provide an efficient route to the 1,3-oxathiolane ring structure, preferably, with substituent groups in place for use in subsequent reactions. Second, the reaction scheme must provide an efficient means to condense the 1,3-oxathiolane ring with a suitably protected base, which, in the case of 3TC is cytosine, and in the case of FTC is 5-fluorocytosine. Third, the reaction must be stereoselective, i.e., it must provide the enantiomer of choice. The substituents on the chiral carbons (the specified purine or pyrimidine base (referred to as the C5 substituent) and CH2OH (referred to as the C2 substituent)) of the 1,3-oxathiolane nucleosides can be either cis (on the same side) or trans (on opposite sides) with respect to the oxathiolane ring system. Both the cis and trans racemates consist of a pair of optical isomers. Hence, each compound has four individual optical isomers. The four optical isomers are represented by the following configurations (when orienting the oxathiolane moiety in a horizontal plane such that the xe2x80x94Sxe2x80x94CH2xe2x80x94 moiety is in back): (1) cis (also referred to as xcex2), with both groups xe2x80x9cupxe2x80x9d, which is the naturally occurring L-cis configuration (2) cis, with both groups xe2x80x9cdownxe2x80x9d, which is the non-naturally occurring xcex2-cis configuration; (3) trans (also referred to as the xcex1-configuration) with the C2 substituent xe2x80x9cupxe2x80x9d and the C5 substituent xe2x80x9cdownxe2x80x9d; and (4) trans with the C2 substituent xe2x80x9cdownxe2x80x9d and the C5 substituent xe2x80x9cupxe2x80x9d. The two cis enantiomers together are referred to as a racemic mixture of xcex2-enantiomers, and the two trans enantiomers are referred to as a racemic mixture of xcex1-enantiomers. In general, it is fairly standard to be able to separate the pair of cis racemic optical isomers from the pair of trans racemic optical isomers. It is a significantly more difficult challenge to separate or otherwise obtain the individual enantiomers of the cis-configuration. For 3TC and FTC, the desired stereochemical configuration is the xcex2-L-isomer.
The numbering scheme for the 1,3-oxathiolane ring is given below. 
Kraus, et al., (xe2x80x9cSynthesis of New 2,5-Disubstituted 1,3-Oxathiolanes. Intermediates in Nucleoside Chemistryxe2x80x9d, Synthesis, pages 1046-1048 (1991)) describe the problems associated with the reaction of an aldehyde of a glyoxylate or glycolic acid with mercaptoacetic acid in toluene in the presence of p-toluenesulfonic acid. Kraus notes that a requirement for the success of this reaction is that glycolic derivatives which exist in the hydrate form have to be converted into the free aldehyde by azeotropic removal of water with toluene before the cyclocondensation. Thereafter, to complete the reduction of both the lactone and carboxylic acid functions, different catalytic reductive reagents had to be employed. Reduction with sodium borohydride failed, and borane-methyl sulfide complex (BMS) was able to reduce only the carboxylic acid function. When the temperature was raised, or a large excess of BMS was used, ring opening occurred leading to polymeric material. Reduction of the 2-carboxy-1,3-oxathiolan-5-one with sodium bis(2-methoxyethoxy)aluminum hydride in toluene gave a mixture of products. Tributyl tin hydride gave no reduction. Finally, when the reduction was performed on the protected lactones, it was not possible to isolate the desired compound, regardless of the catalytic reductive conditions.
Because of these difficulties, Kraus, et al. proposed a reaction that involved the cyclocondensation of anhydrous glyoxylates with 2-mercaptoacetaldehyde diethyl acetal at reflux in toluene to produce 5-ethoxy-1,3-oxathiolane derivatives which could be reduced with BMS to give the corresponding 2-hydroxymethyl-1,3-oxathiolane in 50% yield, which after benzoylation provided a mixture of cis and trans 2-benzoyloxymethyl-5-ethoxy-1,3-oxathiolane. This process is also described in U.S. Pat. No. 5,047,407.
U.S. Pat. No. 5,248,776 discloses a method for the production of enantiomerically pure xcex2-L-1,3-oxathiolane nucleosides from 1,6-thioanyhydro-L-gulose.
U.S. Pat. No. 5,204,466 discloses a route to prepare the 1,3-oxathiolane ring via the reaction of mercaptoacetic acid (thioglycolic acid) with a glycoaldehyde to form 2-(R-oxy)-methyl-5-oxo-1,3-oxathiolane.
U.S. Pat. No. 5,466,806 describes a process for preparing a 2-hydroxymethyl-5-hydroxy-1,3-oxathiolane via the reaction of the dimer of mercaptoacetaldehyde with a compound of the formula RwOCH2CHO under neutral or basic conditions, wherein Rw is a hydroxyl protecting group. See also McIntosh, et al, xe2x80x9c2-Mercaptoaldehyde dimers and 2,5-dihydrothiophenes from 1,2-oxathiolan-5-ones,xe2x80x9d Can. J. Chem. Vol 61, 1872-1875 (1983).
Belleau, et al., disclosed a method to prepare a 1,3-dioxolane nucleoside via the oxidative degradation of L-ascorbic acid. Belleau, et al., xe2x80x9cOxidative Degradation of L-ascorbic Acid Acetals to 2xe2x80x2,3xe2x80x2-Dideoxy-3xe2x80x2-Oxaribofuranosides. Synthesis of Enantiomerically Pure 2xe2x80x2,3xe2x80x2-Dideoxy-3xe2x80x2-Oxacytidine Stereoisomers as Potential Antiviral Agents.,xe2x80x9d Tetrahedron Letters, vol 33, No. 46, 6949-6952 (1992).
U.S. Pat. No. 5,204,466 discloses the preparation of a 1,3-oxathiolane ring via ozonolysis of an allyl ether or ester having the formula CH2xe2x95x90CHCH2OR, in which R is a protecting group, to form a glycoaldehyde having the formula OHCCH2OR, and adding thioglycolic acid to the glycoaldehyde to form a lactone of the formula 2-(R-oxy)-methyl-5-oxo-1,3-oxathiolane.
U.S. Pat. No. 5,204,466 discloses a method to condense a 1,3-oxathiolane with a protected pyrimidine base using tin chloride as a Lewis acid, which provides virtually complete xcex2-stereoselectivity. See also Choi, et al, xe2x80x9cIn Situ Complexation Directs the Stereochemistry of N-Glycosylation in the synthesis of Oxathiolanyl and Dioxolanyl Nucleoside Analogues,xe2x80x9d J. Am Chem. Soc. 1991, 213, 9377-9379. The use of tin chloride creates undesirable residues and side products during the reaction which are difficult to remove.
A number of U.S. patents disclose a process for the preparation of 1,3-oxathiolane nucleosides via the condensation of a 1,3-oxathiolane intermediate that has a chiral ester at the 2-position of the ring, with a protected base in the presence of a silicon-based Lewis acid. The ester at the 2-position must then be reduced to the corresponding hydroxymethyl group to afford the final product. See U.S. Pat. Nos. 5,663,320; 5,864,164; 5,693,787; 5,696,254; 5,744,596; and 5,756,706.
U.S. Pat. No. 5,763,606 discloses a process for producing predominantly cis-2-carboxylic or thiocarboxylic acid 1,3-oxathiolane nucleosides that includes coupling a desired, previously silylated purine or pyrimidine base with a bicyclic intermediate in the presence of a Lewis acid.
U.S. Pat. No. 5,272,151 describes a process for the preparation of 1,3-dioxolane nucleosides that includes reacting a 2-O-protected-5-O-acylated-1,3-dioxolane with an oxygen- or nitrogen-protected purine or pyrimidine base in the presence of a titanium catalyst.
Choi, et al, xe2x80x9cIn Situ Complexation Directs the Stereochemistry of N-Glycosylation in the synthesis of Oxathiolanyl and Dioxolanyl Nucleoside Analogues,xe2x80x9d J. Am Chem. Soc. 1991, 213, 9377-9379, reported that no coupling of the 1,3-oxathiolane with protected pyrimidine base occurs with HgCl2, Et2AlCl, or TiCl2(O-isopropyl)2 (see footnote 2). Choi also reported that the reaction between anomeric 1,3-oxathiolane acetates with silylated cytosine and virtually any common Lewis acid other than tin chloride resulted in the formation of inseparable mixtures of N-glycosylated anomers.
U.S. Pat. No. 5,922,867 discloses a method for preparing a dioxolane nucleoside that includes glycosylating a purine or pyrimidine base with a 2-protected-oxymethyl-4-halo-1,3-dioxolane.
U.S. Pat. No. 5,728,575 claims the method to obtain 3TC and FTC via enzymatic resolution of the 5xe2x80x2-acyl protected racemic nucleoside using pig liver esterase, porcine pancreatic lipase, or subtilisin. U.S. Pat. No. 5,539,116 claims 3TC, the product of the resolution process of the ""575 patent.
U.S. Pat. No. 5,827,727 to Liotta claims the method to obtain 3TC and FTC via stereoselective deamination using cytidine deaminase.
U.S. Pat. No. 5,892,025 to Liotta, et al. claims a method for the resolution of the combination of the enantiomers of cis-FTC by passing the cis-FTC through an acetylated xcex2-cyclodextrin chiral column.
U.S. Pat. No. 5,663,320 claims a process for producing a chiral 1,3-oxathiolane intermediate that includes resolving the racemic intermediate with a chiral auxiliary.
In light of the importance of 1,3-oxathiolane nucleosides in the treatment of human immunodeficiency virus and hepatitis B virus, it is an object of the present invention to provide a process for the production of 1,3-oxathiolane nucleosides which can be used on a manufacturing scale.
Processes for the preparation of 1,3-oxathiolane nucleosides are provided that include efficient methods for the preparation of the 1,3-oxathiolane ring and subsequent condensation of the 1,3-oxathiolane with a pyrimidine or purine base. Using the processes described herein, the compound can be provided as an; isolated enantiomer.
It has been discovered that 2-[R1C(O)OCH2]-1,3-dioxolanyl-5-one can be prepared in high yield by directly reacting an acetal of the formula (R1O)2CHR wherein R is xe2x80x94(CH2xe2x80x94Oxe2x80x94C(O)R1), and each R1 is independently alkyl, aryl, heteroaryl, heterocyclic, alkaryl, alkylheteroaryl, or alkylheterocyclic, or aralkyl, with mercaptoacetic acid in an organic solvent, for example acetonitrile, in the presence of a Lewis or protic acid in an organic solvent with a minimum amount of water. Alternatively, (OH)2CHR or (R1O)(OH)CHR can be used. The acetal can also be used as a mixture of the hemiacetal, the acetal monomer or higher condensation products thereof. By reacting the mercaptoacetic acid directly with the acetal, side products are decreased, which increases product purity and yield of this starting material. The acetal is conveniently produced, as an example, by reacting a diether alcohol with n-butyryl chloride.
(R1O)2CHR or (R1O)(OH)CHR can be prepared by any appropriate route, and for example by either (i) reaction of a compound of the formula OHxe2x80x94CH2xe2x80x94C(H)xe2x95x90C(H)xe2x80x94CH2xe2x80x94OH with R1C(O)Cl to form R1C(O)OCH2C(H)xe2x95x90C(H)CH2OC(O)R1, which is ozonized or otherwise cleaved to form the desired compound; or (ii) reduction of (R1O)2CHC(O)H to form (R1O)2CHCH2OH, which is reacted with ClC(O)R1 to form the desired compound.
In another alternative route, HC(O)CH2OC(O)R1 is reacted with mercaptoacetic acid to form the desired 1,3-oxathiolane ring. HC(O)CH2OC(O)R1 can be prepared by any appropriate route, and for example, by the methods A and B illustrated in FIG. 2.
The 5-(O protecting group)-2-protected hydroxymethyl-1,3-oxathiolane or its 5-acetyloxy derivative can be condensed with a protected silylated pyrimidine or purine base, including cytosine or 5-fluorocytosine, using a Lewis acid such as tin chloride, (Cl)3Ti(isopropoxide), trimethylsilyl triflate, trimethylsilyl iodide, or other Lewis acid known to catalyze the condensation, including those Lewis acids described in U.S. Pat. Nos. 5,663,320; 5,864,164; 5,693,787; 5,696,254; 5,744,596; and 5,756,706 to provide the corresponding nucleoside with high xcex2-selectivity. It is surprising that (Cl)3Ti(isopropoxide) is useful as a catalyst for condensation of the 1,3-oxathiolane with the protected base, given that it has been reported that no coupling of the 1,3-oxathiolane with protected pyrimidine base occurs with HgCl2, Et2AlCl, or TiCl2(O-isopropyl)2.
In an alternative embodiment, glycolic acid is substituted for mercaptoacetic acid in the presence of a Lewis acid to form the corresponding 1,3-dioxolane, which can be condensed with a purine or pyrimidine base to provide a 1,3-dioxolane nucleoside. It is preferred to conduct the cyclocondensation of an acetal (or aldehyde) with glycolic acid in the presence of a Lewis acid such as boron trifluoride diethyl etherate rather than a protic acid such as p-toluenesulfonic acid.
It has also been discovered that a 1,3-oxathiolane nucleoside can be produced by: (i) preparing a 5-halo-2-protected-oxymethyl-1,3-oxathiolane; and (ii) reacting the 5-halo-2-protected-oxymethyl-1,3-oxathiolane with a protected purine or pyrimidine base at low temperature, and preferably below 25 degrees Celcius, and more preferably below 10 degrees Celcius. It was surprising that the condensation reaction can be carried out effectively without the aid of a Lewis acid. In a preferred embodiment, the halogen in the 5-position of the oxathiolane is a chloro substituent. The reaction typically produces a mixture of xcex2 and xcex1 anomers that must be separated. The xcex2 anomer typically is produced in excess over the a anomer. The separation of xcex2 and xcex1 anomers can be effected by any known method, including fractional crystallization, chromatography (achiral or chiral), or the preparation and separation of diastereomeric derivatives. In one embodiment, a racemic 5-acylated-2-protected-oxymethyl-1,3-oxathiolane is chlorinated at low temperature (e.g., 0 degrees Celcius), and then condensed with a protected base such as 5-fluorocytosine or cytosine, to produce a mixture of diastereomers (with the xcex2 compound typically in substantial excess). In another embodiment, a chiral 5-acylated-2-protected-oxymethyl-1,3-oxathiolane is chlorinated and then reacted with a protected base. Any 5-acylated-2-protected-oxymethyl-1,3-oxathiolane can be used that provides the desired product. Nonlimiting examples of appropriate acyl moieties include, but are not limited to acetate, propionate, butyrate, benzoate, p-methoxybenzoate, and p-(t-butyl)-benzoate. The halogenaton reaction can be carried out in any useful organic solvent, including toluene, chloroform, acetic acid, tetrahydrofuran, ether, benzene, etc. The anomeric ratio of xcex1 to xcex2 produced in the condensation reaction can be affected by the solvent selected for use in the reaction. One can easily test various organic solvents to select that solvent which provides the optimal yield of the desired product.